The major goal of this project is to develop synthetic small molecules that have many of the properties of gene-specific transcriptional activators. These molecules would consist of fragments capable of recognizing DNA sequence-specifically fused to moieties capable of binding to co- activators or co-repressors, which serve as regulators of the DNA polymerase II transcriptional machinery. If cell-permeable synthetic molecules of this type could be constructed, they could be used to up- or down-regulate the expression of any gene desired. This would constitute an extraordinary powerful tool with which to manipulate cellular metabolism. In this proposal, we focus on a few particular gene targets of importance in the potential treatment of obesity and diabetes. Work by our collaborators, Roger Unger and Christ Newgard, has strongly suggested that up-regulation of a number of genes which are involved in fat-burning reactions would be of great clinical benefit in the treatment of obesity and some forms of diabetes. Since the expression of many or all of these genes is stimulated by the transcription factor PPAR-a, the specific goal of this proposal will be to develop compounds capable of up-regulating the expression of this gene, which is then expected to stimulate the fat- burning pathway through secondary activation of the genes encoding the metabolic enzymes. An alternative strategy would be to create several different molecules able to recognize each of the promoters of the metabolic genes, and this would constitute a fallback plan in case of difficulty in targeting PPAR-alpha is encountered. In the first phase of this work, cells will be engineered to express co- activator proteins with a tag that will allow the use of known small molecules as activation domain mimics. These known molecules will be fused synthetically to either PNAs or, later, polyamides of the typed developed by Dervan and co-workers. The resulting chimeras will be tested in vitro and in cell culture for the ability to activate transcription of the PPAR-a gene specifically. In the second phase of the project, combinatorial chemistry methods will be employed to identify molecules able to bind specifically to native, unmodified co-activators. Chimeras of these molecules fused to the appropriate sequence-specific DNA-binding moiety, should allow the activation of PPAR-alpha or other target genes, in wild-type cells.